title: Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.
abstract: The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles.
authors.affiliation: Department of Pharmacology and Medicine, Division of Pulmonary and Critical Care, Rush University, Chicago, Illinois, United States of America.
authors.forename: Sanda
authors.initials: S
authors.lastname: Predescu
authors.forename: Ivana
authors.initials: I
authors.lastname: Knezevic
authors.forename: Cristina
authors.initials: C
authors.lastname: Bardita
authors.forename: Radu Florin
authors.initials: RF
authors.lastname: Neamu
authors.forename: Viktor
authors.initials: V
authors.lastname: Brovcovych
authors.forename: Dan
authors.initials: D
authors.lastname: Predescu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002199:Capillary Permeability / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002518:Ceramides / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism
chemical_list: D002518:Ceramides; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D052250:Nitric Oxide Synthase Type III; D013108:Sphingomyelin Phosphodiesterase